Lawrence L. Spriet

Repeated transient mRNA bursts precede increases in transcriptional and mitochondrial proteins during training in human skeletal muscle

Exercise training induces mitochondrial biogenesis, but the time course of molecular sequelae that accompany repetitive training stimuli remains to be determined in human skeletal muscle. Therefore, throughout a seven‐session, high‐intensity interval training period that increased (12%), we examined the time course of responses of (a) mitochondrial biogenesis and fusion and fission proteins, and (b) selected transcriptional and mitochondrial mRNAs and proteins in human muscle. Muscle biopsies were obtained 4 and 24 h after the 1st, 3rd, 5th and 7th training session. PGC‐1α mRNA was increased >10‐fold 4 h after the 1st session and returned to control within 24 h. This ‘saw‐tooth’ pattern continued until the 7th bout, with smaller increases after each bout. In contrast, PGC‐1α protein was increased 24 h after the 1st bout (23%) and plateaued at +30–40% between the 3rd and 7th bout. Increases in PGC‐1β mRNA and protein were more delayed and smaller, and did not persist. Distinct patterns of increases were observed in peroxisome proliferator‐activated receptor (PPAR) α and γ protein (1 session), PPAR β/δ mRNA and protein (5 sessions) and nuclear respiratory factor‐2 protein (3 sessions) while no changes occurred in mitochondrial transcription factor A protein. Citrate synthase (CS) and β‐HAD mRNA were rapidly increased (1 session), followed 2 sessions later (session 3) by increases in CS and β‐HAD activities, and mitochondrial DNA. Changes in COX‐IV mRNA (session 3) and protein (session 5) were more delayed. Training also increased mitochondrial fission proteins (fission protein‐1, >2‐fold; dynamin‐related protein‐1, 47%) and the fusion protein mitofusin‐1 (35%) but not mitofusin‐2. This study has provided the following novel information: (a) the training‐induced increases in transcriptional and mitochondrial proteins appear to result from the cumulative effects of transient bursts in their mRNAs, (b) training‐induced mitochondrial biogenesis appears to involve re‐modelling in addition to increased mitochondrial content, and (c) the ‘transcriptional capacity’ of human muscle is extremely sensitive, being activated by one training bout.

High-intensity aerobic interval training increases fat and carbohydrate metabolic capacities in human skeletal muscle

High-intensity aerobic interval training (HIIT) is a compromise between time-consuming moderate-intensity training and sprint-interval training requiring all-out efforts. However, there are few data regarding the ability of HIIT to increase the capacities of fat and carbohydrate oxidation in skeletal muscle. Using untrained recreationally active individuals, we investigated skeletal muscle and whole-body metabolic adaptations that occurred following 6 weeks of HIIT (~1 h of 10 x 4 min intervals at ~90% of peak oxygen consumption (VO2 peak), separated by 2 min rest, 3 d.week-1). A VO2 peak test, a test to exhaustion (TE) at 90% of pre-training VO2 peak, and a 1 h cycle at 60% of pre-training VO2 peak were performed pre- and post-HIIT. Muscle biopsies were sampled during the TE at rest, after 5 min, and at exhaustion. Training power output increased by 21%, and VO2 peak increased by 9% following HIIT. Muscle adaptations at rest included the following: (i) increased cytochrome c oxidase IV content (18%) and maximal activities of the mitochondrial enzymes citrate synthase (26%), beta-hydroxyacyl-CoA dehydrogenase (29%), aspartate-amino transferase (26%), and pyruvate dehydrogenase (PDH; 21%); (ii) increased FAT/CD36, FABPpm, GLUT 4, and MCT 1 and 4 transport proteins (14%-30%); and (iii) increased glycogen content (59%). Major adaptations during exercise included the following: (i) reduced glycogenolysis, lactate accumulation, and substrate phosphorylation (0-5 min of TE); (ii) unchanged PDH activation (carbohydrate oxidation; 0-5 min of TE); (iii) ~2-fold greater time during the TE; and (iv) increased fat oxidation at 60% of pre-training VO2 peak. This study demonstrated that 18 h of repeated high-intensity exercise sessions over 6 weeks (3 d.week-1) is a powerful method to increase whole-body and skeletal muscle capacities to oxidize fat and carbohydrate in previously untrained individuals.

Regulation of skeletal muscle glycogen phosphorylase and PDH during maximal intermittent exercise.

The time course for the activation of glycogen phosphorylase (Phos) and pyruvate dehydrogenase (PDH) and their allosteric regulators was determined in human skeletal muscle during repeated bouts of maximal exercise. Six subjects completed three 30-s bouts of maximal isokinetic cycling separated by 4-min recovery periods. Muscle biopsies were taken at rest and at 6, 15, and 30 s of exercise during bouts 1 and 3. Phos was rapidly activated within the first 6 s of bout 1 from 12% at rest to 47% at 6 s. The activation of PDH increased from 14% at rest to 48% at 6 s and 95% at 15 s of bout 1. Phos reverted back to basal values at the end of the first bout, whereas PDH remained fully activated. In contrast, in the third bout, PDH was 42% at rest and was activated more rapidly and was nearly completely activated by 6 s, whereas Phos remained at basal levels (range 14-20%). Lactate accumulation was marked in the first bout and increased progressively from 2.7 to 76.1 mmol/kg dry wt with no further increase in bout 3. Glycogen utilization was also marked in the first bout and was negligible in bout 3. The rapid activation of Phos and slower activation of PDH in bout 1was probably due to Ca2+ release from the sarcoplasmic reticulum. Lactate accumulation appeared to be due to an imbalance of the relative activities of Phos and PDH. The increase in H+ concentration may have served to reduce pyruvate production by inhibiting Phos transformation and may have simultaneously activated PDH in the third bout such that there was a better matching between pyruvate production and oxidation and minimal lactate accumulation. As each bout progressed and with successive bouts, there was a decreasing ability to stimulate substrate phosphorylation through phosphocreatine hydrolysis and glycolysis and a shift toward greater reliance on oxidative phosphorylation.

Mitochondrial long chain fatty acid oxidation, fatty acid translocase/CD36 content and carnitine palmitoyltransferase I activity in human skeletal muscle during aerobic exercise

Mitochondrial fatty acid transport is a rate‐limiting step in long chain fatty acid (LCFA) oxidation. In rat skeletal muscle, the transport of LCFA at the level of mitochondria is regulated by carnitine palmitoyltransferase I (CPTI) activity and the content of malonyl‐CoA (M‐CoA); however, this relationship is not consistently observed in humans. Recently, fatty acid translocase (FAT)/CD36 was identified on mitochondria isolated from rat and human skeletal muscle and found to be involved in LCFA oxidation. The present study investigated the effects of exercise (120 min of cycling at ∼60%) on CPTI palmitoyl‐CoA and M‐CoA kinetics, and on the presence and functional significance of FAT/CD36 on skeletal muscle mitochondria. Whole body fat oxidation rates progressively increased during exercise (P < 0.05), and concomitantly M‐CoA inhibition of CPTI was progressively attenuated. Compared to rest, 120 min of cycling reduced (P < 0.05) the inhibition of 0.7, 2, 5 and 10 μm M‐CoA by 16%, 21%, 30% and 34%, respectively. Whole body fat oxidation and palmitate oxidation rates in isolated mitochondria progressively increased (P < 0.05) during exercise, and were positively correlated (r= 0.78). Mitochondrial FAT/CD36 protein increased by 63% (P < 0.05) during exercise and was significantly (P < 0.05) correlated with mitochondrial palmitate oxidation rates at all time points (r= 0.41). However, the strongest (P < 0.05) correlation was observed following 120 min of cycling (r= 0.63). Importantly, the addition of sulfo‐N‐succimidyloleate, a specific inhibitor of FAT/CD36, reduced mitochondrial palmitate oxidation to ∼20%, indicating FAT/CD36 is functionally significant with respect to LCFA oxidation. We hypothesize that exercise‐induced increases in fatty acid oxidation occur as a result of an increased ability to transport LCFA into mitochondria. We further suggest that decreased CPTI M‐CoA sensitivity and increased mitochondrial FAT/CD36 protein are both important for increasing whole body fatty acid oxidation during prolonged exercise.

Intramuscular triacylglycerol, glycogen and acetyl group metabolism during 4 h of moderate exercise in man

This study investigated intramuscular triacylglycerol (IMTG) and glycogen utilisation, pyruvate dehydrogenase activation (PDHa) and acetyl group accumulation during prolonged moderate intensity exercise. Seven endurance‐trained men cycled for 240 min at 57 % maximal oxygen consumption (V̇O2,max) and duplicate muscle samples were obtained at rest and at 10, 120 and 240 min of exercise. We hypothesised that IMTG utilisation would be augmented during 2‐4 h of exercise, while PDHa would be decreased secondary to reduced glycogen metabolism. IMTG was measured on both muscle samples at each time point and the coefficient of variation was 12.3 ± 9.4 %. Whole body respiratory exchange ratio (RER) decreased from 0.89 ± 0.01 at 30 min to 0.83 ± 0.01 at 150 min and remained low throughout exercise. Plasma glycerol and free fatty acids (FFAs) had increased compared with rest at 90 min and progressively increased until exercise cessation. Although plasma glucose tended to decrease with exercise, this was not significant. IMTG was reduced at 120 min compared with rest (0 min, 15.6 ± 0.8 mmol kg−1 d.m.; 120 min, 12.8 ± 0.7 mmol kg−1 d.m.) but no further reduction in IMTG was observed at 240 min. Muscle glycogen was 468 ± 49 mmol kg−1 d.m. at rest and decreased at 120 min and again at 240 min (217 ± 48 and 144 + 47 mmol kg−1 d.m.). PDHa increased above rest at 10 and 120 min, but decreased at 240 min, which coincided with reduced whole body carbohydrate oxidation. Muscle pyruvate and ATP were unchanged with exercise. Acetyl CoA increased at 10 min and remained elevated throughout exercise. Acetylcarnitine increased at exercise onset but returned to resting values by 240 min. Contrary to our first hypothesis, significant utilisation of IMTG occurred during the first 2 h of moderate exercise but not during hours 2‐4. The reduced utilisation of intramuscular fuels during the last 120 min was offset by greater FFA delivery and oxidation. Consistent with the second hypothesis, PDHa decreased late in moderate exercise and closely matched the estimates of lower carbohydrate flux. Although the factor underlying the PDHa decrease was not apparent, reduced pyruvate provision secondary to diminished glycolytic flux is the most likely mechanism.

Nutritional modulation of training-induced skeletal muscle adaptations

Skeletal muscle displays remarkable plasticity, enabling substantial adaptive modifications in its metabolic potential and functional characteristics in response to external stimuli such as mechanical loading and nutrient availability. Contraction-induced adaptations are determined largely by the mode of exercise and the volume, intensity, and frequency of the training stimulus. However, evidence is accumulating that nutrient availability serves as a potent modulator of many acute responses and chronic adaptations to both endurance and resistance exercise. Changes in macronutrient intake rapidly alter the concentration of blood-borne substrates and hormones, causing marked perturbations in the storage profile of skeletal muscle and other insulin-sensitive tissues. In turn, muscle energy status exerts profound effects on resting fuel metabolism and patterns of fuel utilization during exercise as well as acute regulatory processes underlying gene expression and cell signaling. As such, these nutrient-exercise interactions have the potential to activate or inhibit many biochemical pathways with putative roles in training adaptation. This review provides a contemporary perspective of our understanding of the molecular and cellular events that take place in skeletal muscle in response to both endurance and resistance exercise commenced after acute and/or chronic alterations in nutrient availability (carbohydrate, fat, protein, and several antioxidants). Emphasis is on the results of human studies and how nutrient provision (or lack thereof) interacts with specific contractile stimulus to modulate many of the acute responses to exercise, thereby potentially promoting or inhibiting subsequent training adaptation.

Adrenaline increases skeletal muscle glycogenolysis, pyruvate dehydrogenase activation and carbohydrate oxidation during moderate exercise in humans

1 To evaluate the role of adrenaline in regulating carbohydrate metabolism during moderate exercise, 10 moderately trained men completed two 20 min exercise bouts at 58 ± 2 % peak pulmonary oxygen uptake (V̇O2,peak). On one occasion saline was infused (CON), and on the other adrenaline was infused intravenously for 5 min prior to and throughout exercise (ADR). Glucose kinetics were measured by a primed, continuous infusion of 6,6‐[2H]glucose and muscle samples were obtained prior to and at 1 and 20 min of exercise. 2 The infusion of adrenaline elevated (P < 0.01) plasma adrenaline concentrations at rest (pre‐infusion, 0.28 ± 0.09; post‐infusion, 1.70 ± 0.45 nmol l−1; means ±s.e.m.) and this effect was maintained throughout exercise. Total carbohydrate oxidation increased by 18 % and this effect was due to greater skeletal muscle glycogenolysis (P < 0.05) and pyruvate dehydrogenase (PDH) activation (P < 0.05, treatment effect). Glucose rate of appearance was not different between trials, but the infusion of adrenaline decreased (P < 0.05, treatment effect) skeletal muscle glucose uptake in ADR. 3 During exercise muscle glucose 6‐phosphate (G‐6‐P) (P = 0.055, treatment effect) and lactate (P < 0.05) were elevated in ADR compared with CON and no changes were observed for pyruvate, creatine, phosphocreatine, ATP and the calculated free concentrations of ADP and AMP. 4 The data demonstrate that elevated plasma adrenaline levels during moderate exercise in untrained men increase skeletal muscle glycogen breakdown and PDH activation, which results in greater carbohydrate oxidation. The greater muscle glycogenolysis appears to be due to increased glycogen phosphorylase transformation whilst the increased PDH activity cannot be readily explained. Finally, the decreased glucose uptake observed during exercise in ADR is likely to be due to the increased intracellular G‐6‐P and a subsequent decrease in glucose phosphorylation.

Regulation of skeletal muscle glycogen phosphorylase and PDH at varying exercise power outputs

This study investigated the transformational and posttransformational control of skeletal muscle glycogen phosphorylase and pyruvate dehydrogenase (PDH) at three exercise power outputs [35, 65, and 90% of maximal oxygen uptake (VO2 max)]. Seven untrained subjects cycled at one power output for 10 min on three separate occasions, with muscle biopsies at rest and 1 and 10 min of exercise. Glycogen phosphorylase in the more active (a) form was not significantly different at any time across power outputs (21. 4-29.6%), with the exception of 90%, where it fell significantly to 15.3% at 10 min. PDH transformation increased significantly from rest (average 0.53 mmol . kg wet muscle-1 . min-1) to 1 min of exercise as a function of power output (1.60 +/- 0.26, 2.77 +/- 0.29, and 3.33 +/- 0.31 mmol . kg wet muscle-1 . min-1 at 35, 65, and 90%, respectively) with a further significant increase at 10 min (4.45 +/- 0.35) at 90% VO2 max. Muscle lactate, acetyl-CoA, acetylcarnitine, and free ADP, AMP, and Pi were unchanged from rest at 35% VO2 max but rose significantly at 65 and 90%, with accumulations at 90% being significantly higher than 65%. The results of this study indicate that glycogen phosphorylase transformation is independent of increasing power outputs, despite increasing glycogenolytic flux, suggesting that flux through glycogen phosphorylase is matched to the demand for energy by posttransformational factors, such as free Pi and AMP. Conversely, PDH transformation is directly related to the increasing power output and the calculated flux through the enzyme. The rise in PDH transformation is likely due to increased Ca2+ concentration and/or increased pyruvate. These results demonstrate that metabolic signals related to contraction and the energy state of the cell are sensitive to the exercise intensity and coordinate the increase in carbohydrate use with increasing power output.

An enzymatic approach to lactate production in human skeletal muscle during exercise.

PURPOSE This paper examines the production of lactate in human skeletal muscle over a range of power outputs (35-250% VO2max) from an enzymatic flux point of view. The conversion of pyruvate and NADH to lactate and NAD in the cytoplasm of muscle cells is catalyzed by the near-equilibrium enzyme lactate dehydrogenase (LDH). As flux through LDH is increased by its substrates, pyruvate and NADH, the factors governing the production of these substrates will largely dictate how much lactate is produced at any exercise power output. In an attempt to understand lactate production, flux rates through the enzymes that regulate glycogenolysis/glycolysis, the transfer of cytoplasmic reducing equivalents into the mitochondria, and the various fates of pyruvate have been measured or estimated. RESULTS At low power outputs, the rates of pyruvate and NADH production in the cytoplasm are low, and pyruvate dehydrogenase (PDH) and the shuttle system enzymes (SS) metabolize the majority of these substrates, resulting in little or no lactate production. At higher power outputs (65, 90, and 250% VO2max), the mismatch between the ATP demand and aerobic ATP provision at the onset of exercise increases as a function of intensity, resulting in increasing accumulations of the glycogenolytic/glycolytic activators (free ADP, AMP, and Pi). The resulting glycolytic flux, and NADH and pyruvate production, is progressively greater than can be handled by the SS and PDH, and lactate is produced at increasing rates. Lactate production during the onset of exercise and 10 min of sustained aerobic exercise may be a function of adjustments in the delivery of O2 to the muscles, adjustments in the activation of the aerobic ATP producing metabolic pathways and/or substantial glycogenolytic/glycolytic flux through a mass action effect.

Skeletal muscle malonyl-CoA content at the onset of exercise at varying power outputs in humans

To investigate the regulation of intramuscular fuel selection, we measured the malonyl-CoA (M-CoA) content in human skeletal muscle at three exercise power outputs [35, 65, and 90% maximal rate of O2 consumption (VO2 max)]. Four males and four females cycled for 10 min at one power output on three separate occasions with muscle biopsies sampled at rest and at 1 and 10 min. The respiratory exchange ratio was 0.84 +/- 0.03, 0.92 +/- 0.02, and >1.0 at 35, 65 and 90% VO2 max, respectively. Muscle lactate content increased and phosphocreatine content decreased as a function of power output. Pyruvate dehydrogenase a activity increased from 0.40-0.64 mmol . kg wet muscle-1 . min-1 at rest to 1.57 +/- 0.28, 2.80 +/- 0.41, and 3. 28 +/- 0.27 mmol . kg wet muscle-1 . min-1 after 1 min of cycling at the three power outputs, respectively. Mean resting M-CoA contents were similar at all power outputs (1.85-1.98 micromol/kg dry muscle). During exercise at 35% VO2 max, M-CoA decreased from rest at 1 min (1.85 +/- 0.29 to 1.20 +/- 0.12 micromol/kg dry muscle) but returned to rest level by 10 min (1.86 +/- 0.25 micromol/kg dry muscle). M-CoA content did not decrease during cycling at 65% VO2 max. At 90% VO2 max, M-CoA did not increase despite significant acetyl-CoA accumulation (the substrate for M-CoA synthesis). The data suggest that a decrease in M-CoA content is not required for the increase in free fatty acid uptake and oxidation that occurs during exercise at 35 and 65% VO2 max. Furthermore, M-CoA content does not increase during exercise at 90% VO2 max and does not contribute to the lower rate of fat oxidation at this power output.To investigate the regulation of intramuscular fuel selection, we measured the malonyl-CoA (M-CoA) content in human skeletal muscle at three exercise power outputs [35, 65, and 90% maximal rate of O2 consumption (V˙o 2 max)]. Four males and four females cycled for 10 min at one power output on three separate occasions with muscle biopsies sampled at rest and at 1 and 10 min. The respiratory exchange ratio was 0.84 ± 0.03, 0.92 ± 0.02, and >1.0 at 35, 65 and 90%V˙o 2 max, respectively. Muscle lactate content increased and phosphocreatine content decreased as a function of power output. Pyruvate dehydrogenase a activity increased from 0.40-0.64 mmol ⋅ kg wet muscle-1 ⋅ min-1at rest to 1.57 ± 0.28, 2.80 ± 0.41, and 3.28 ± 0.27 mmol ⋅ kg wet muscle-1 ⋅ min-1after 1 min of cycling at the three power outputs, respectively. Mean resting M-CoA contents were similar at all power outputs (1.85-1.98 μmol/kg dry muscle). During exercise at 35%V˙o 2 max, M-CoA decreased from rest at 1 min (1.85 ± 0.29 to 1.20 ± 0.12 μmol/kg dry muscle) but returned to rest level by 10 min (1.86 ± 0.25 μmol/kg dry muscle). M-CoA content did not decrease during cycling at 65%V˙o 2 max. At 90%V˙o 2 max, M-CoA did not increase despite significant acetyl-CoA accumulation (the substrate for M-CoA synthesis). The data suggest that a decrease in M-CoA content is not required for the increase in free fatty acid uptake and oxidation that occurs during exercise at 35 and 65%V˙o 2 max. Furthermore, M-CoA content does not increase during exercise at 90%V˙o 2 max and does not contribute to the lower rate of fat oxidation at this power output.